The biological material listed below has been deposited with the American Type Culture Center (10801 University Blvd., Manassas, Va.):
The most common bacterial pathogens of plants colonize the apoplast, and from that location outside of the walls of living cells they incite a variety of diseases in most cultivated plants (Alfano et al., xe2x80x9cBacterial Pathogens in Plants: Life Up Against the Wall,xe2x80x9d Plant Cell 8:1683-1698 (1996)). The majority of these are Gram-negative bacteria in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia. Most are host specific and will elicit the hypersensitive response (xe2x80x9cHRxe2x80x9d) in nonhosts. The HR is a rapid, programmed death of plant cells in contact with the pathogen. Some of the defense responses associated with the HR are localized at the periphery of plant cells at the site of bacterial contact, but what actually stops bacterial growth is not known (Brown et al., xe2x80x9chrp genes in Xanthomonas campestris pv. vesicatoria Determine Ability to Suppress Papilla Deposition in Pepper Mesophyll Cells,xe2x80x9d MPMI 8:825-836 (1995); Young et al., xe2x80x9cChanges in the Plasma Membrane Distribution of Rice Phospholipase D During Resistant Interactions With Xanthomonas oryzae pv. oryzae,xe2x80x9d Plant Cell 8:1079-1090 (1996); Bestwick et al., xe2x80x9cLocalization of Hydrogen Peroxide Accumulation During the Hypersensitive Reaction of Lettuce Cells to Pseudomonas syringae pv. phaseolicola,xe2x80x9d Plant Cell 9:209-221 (1997)). Pathogenesis in host plants, in contrast, involves prolonged bacterial multiplication, spread to surrounding tissues, and the eventual production of macroscopic symptoms characteristic of the disease. Although these bacteria are diverse in their taxonomy and pathology, they all possess hrp genes which direct their ability to elicit the HR in nonhosts or to be pathogenic (and parasitic) in hosts (Lindgren, xe2x80x9cThe Role of hrp Genes During Plant-Bacterial Interactions,xe2x80x9d Annu. Rev. Phytopathol. 35:129-152 (1997)). The hrp genes encode a type III protein secretion system that appears to be capable of delivering Avr (avirulence) proteins across the walls and plasma membranes of living plant cells (Alfano et al., xe2x80x9cThe Type III (Hrp) Secretion Pathway of Plant Pathogenic Bacteria: Trafficking Harpins, Avr Proteins, and Death,xe2x80x9d J. Bacteriol. 179:5655-5662 (1997), which is hereby incorporated by reference). The Avr proteins are so named because they can betray the parasite to the R gene-encoded surveillance system of plants, thereby triggering the HR (Vivian et al., xe2x80x9cAvirulence Genes in Plant-Pathogenic Bacteria: Signals or Weapons?,xe2x80x9d Microbiology 143:693-704 (1997); Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annul. Rev. Phytopathol. 34:153-179 (1996)). But Avr-like proteins also appear to be key to parasitism in compatible host plants, where the parasite proteins are undetected and the HR is not triggered. Thus, bacterial avirulence and pathogenicity are interrelated phenomena and explorations of HR elicitation are furthering our understanding of parasitic mechanisms.
Despite the emerging importance of Avr proteins, there is no direct evidence that they travel the Hrp pathway, there is no knowledge of their function in virulence, it appears likely that only a subset of those that are produced by typical host-specific pathogens have been identified, and there is no evidence that they are produced at all by host-promiscuous pathogens. The evidence that Avr proteins are transferred by the Hrp pathway into plants is most complete, although still indirect, with Pseudomonas syringae AvrB and AvrPto proteins. Nonpathogenic Escherichia coli and Pseudomonas fluorescens cells that harbor the functional cluster of Pseudomonas syringae hrp genes carried on cosmid pHIR11 can elicit an HR that is dependent on both the type III secretion system and either AvrB or AvrPto (Gopalan et al., xe2x80x9cExpression of the Pseudomonas Syringae Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,xe2x80x9d Plant Cell 8:1095-1105 (1996); Pirhonen et al., xe2x80x9cPhenotypic Expression of Pseudomonas Syringae avr Genes in E. coli is Linked to the Activities of the hrp-encoded Secretion System,xe2x80x9d MPMI 9:252-260 (1996)). Both Avr proteins trigger an R gene-dependent HR when transiently expressed inside plant cells (Gopalan et al., xe2x80x9cExpression of the Pseudomonas Syringae Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,xe2x80x9d Plant Cell 8:1095-1105 (1996)) and the interaction of AvrPto and Pto in the yeast two-hybrid system correlates with biological activity (Tang et al., Science 274:2060 (1996); Scofield et al., Science 274:2063-2065 (1996)). However, neither Pseudomonas syringae, Escherichia coli (pHIR11), nor Pseudomonas fluorescens (pHIR11) secrete AvrB or AvrPto in culture, presumably because these proteins travel the type III pathway directly into host cells and only upon host cell contact, as with the Yop virulence proteins of Yersinia spp. (Gopalan et al., xe2x80x9cExpression of the Pseudomonas syringae Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and Pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,xe2x80x9d Plant Cell 8:1095-1105 (1996); Cornelis et al., xe2x80x9cThe Yersinia Yop Regulon: A Bacterial System for Subverting Eukaryotic Cells,xe2x80x9d Mol. Microbiol. 23:861-867 (1997)). Other known Avr proteins have been observed only in the bacterial cytoplasm (Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annu. Rev. Phytopathol. 34:153-179 (1996); Knoop et al., xe2x80x9cExpression of the Avirulence Gene avrBs3 from Xanthomonas campestris pv. vesicatoria is not Under the Control of hrp Genes and is Independent of Plant Factors,xe2x80x9d J. Bacteriol. 173:7142-7150 (1991); Puri et al., xe2x80x9cExpression of avrPphB, an Avirulence Gene from Pseudomonas Syringae pv. Phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean,xe2x80x9d MPMI 10:247-256 (1997)).
Many proteins and polypeptides, including hormones and enzymes, are in high demand for pharmacological and industrial use. Once the gene encoding a desired protein or polypeptide has been isolated, the protein can be produced readily through fermentation in rapidly growing bacteria. Escherichia coli is used most commonly for large-scale protein production. Current technology enables the production of relatively large intracellular concentrations of the desired proteins or polypeptides. Extraction of the desired protein or polypeptide from the bacterial cells requires lysing of the cell membrane. After lysing the cell membrane, the desired protein or polypeptide is contaminated with other proteins and, therefore, subject to degradation. The resulting contamination requires significant purification to obtain the isolated protein or polypeptide and degradation of the desired protein or polypeptide limits the obtainable yield.
In addition to fermentation technologies for production of proteins or polypeptides, gene therapy involving transgenic plants is emerging as an important tool for enhancing agricultural productivity and reducing disease losses. For example, transgenic plants expressing bacterial and viral proteins are now used for herbicide tolerance and resistance to viral diseases, respectively. Because of the ease with which foreign proteins can be expressed in most major crops, it is feasible to bioprospect for proteins that will alter plant metabolism to enhance productivity and prevent losses due to pests.
Phytopathogenic bacteria contain a reservoir of genes encoding proteins that have evolved to be biologically active inside plants. Although poorly understood at this point, these proteins are likely to alter plant growth and development, affect fundamental cellular processes common to all higher organisms, including both plants and animals, and/or interact with defense mechanisms. The reservoir of these genes is potentially large, but only a relatively small number have been identified among all of the phytopathogenic bacteria, because identifying them has been dependent upon inefficient procedures involving transgenic pathogens, plant inoculations, and plant reactions.
Thus, it would be beneficial to obtain a recombinant construct and expression system which overcomes these and other deficiencies in the art, particularly the ability to produce a recombinant host organism capable of expressing and secreting Avr and/or other desired proteins or polypeptides into their environment (i.e., culture medium).
One aspect of the present invention relates to a DNA construct that contains a first DNA molecule encoding a functional type III secretion system, a promoter, and a second DNA molecule encoding a protein or polypeptide capable of being secreted by the type III secretion system. The second DNA molecule is operably coupled to the promoter so that upon introduction of the DNA construct into a host cell, the encoded protein or polypeptide and the type III secretion system are expressed and the encoded protein or polypeptide is secreted. Also disclosed are host cells and expression systems that contain the DNA construct, as well as a method of secreting a protein or polypeptide into the environment of a host cell which employs the DNA construct.
Another aspect of the present invention relates to a system that includes a (i) first DNA construct having a first DNA molecule encoding a functional type III secretion system and (ii) a second DNA construct having a promoter operably coupled to a second DNA molecule encoding a protein or polypeptide capable of being secreted by the type III secretion system. Upon introduction of the first and second DNA constructs into a host cell, the encoded protein or polypeptide and the type III secretion system are expressed and the encoded protein or polypeptide is secreted. Also disclosed are host cells and expression systems that contain the system of DNA constructs, as well as a method of secreting a protein or polypeptide into the environment of a host cell which employs the system of DNA constructs.
A further aspect of the present invention relates to a method of isolating a protein or polypeptide. This method is performed by providing a recombinant host cell that contains (i) a first DNA molecule encoding a functional type III secretion system and (ii) a second, heterologous DNA molecule having a promoter operably coupled to a nucleic acid sequence encoding a protein or polypeptide capable of being secreted by the type III secretion system. The recombinant host cell is introduced into a culture medium, wherein the encoded protein or polypeptide and the type III secretion system are expressed and the encoded protein or polypeptide is secreted into the culture medium. Subsequently, the encoded protein or polypeptide is isolated from the culture medium.
Still another aspect of the present invention relates to a method of identifying a gene encoding a potential effector protein or polypeptide. This method of the invention is performed by providing a host cell that contains a DNA molecule encoding a functional type III secretion system. Next, a candidate gene encoding a protein or polypeptide is inserted into the host cell under conditions effective to express the encoded protein or polypeptide. Finally, it is determined whether the encoded protein or polypeptide is secreted by the recombinant host cell, wherein secretion of the encoded protein or polypeptide indicates that the gene encodes a potential effector protein or polypeptide.
Since the DNA constructs of the present invention enable expression and secretion of proteins by recombinant host cells, it is possible to employ these recombinant host cells in a fermentation system which enables efficient production of a desired protein or polypeptide that can be purified at high yield and at minimal expense compared to existing fermentation/purification procedures. Moreover, the constructs of the present invention can be employed to bioprospect for potential effector proteins or polypeptides, which by virtue of their expression and secretion by a recombinant host cell expressing a type III secretion system, become likely candidates as effector proteins. This method of screening for potential effector protein is novel and much more systematic and efficient than prior methods.